Defects in the maintenance of stem cells can result in loss of the ability of tissues to regenerate or, conversely, in dysregulated growth and cancer. How the stem cell resists losing its regenerative qualities while retaining control of proliferation remains poorly understood. The melanocyte stem cell (MSC) is an ideal model for the study of adult stem cells in general. Either the apoptosis or differentiation of this cell type can cause hair graying, and mutations in MITF, which encodes the master regulator of melanocyte development, have been shown to cause hair graying in mice. Mutations in TFAP2A (Transcription Factor Activating-enhancer binding Protein 2 alpha) cause branchio-oculo-facial syndrome (BOFS), which includes premature hair graying. However, the mechanisms whereby TFAP2 proteins contribute to the relevant regulatory networks in MSCs remain unclear. Based on extensive preliminary data from zebrafish, mice, and cultured human melanocytes, our overall hypothesis is that AP-2 activity regulates pluripotency, growth and differentiation in melanocytes and MSCs, working in parallel with MITF and regulated by KCTD15-mediated sumoylation. In Aim 1 we will test the hypothesis that that cell-autonomous TFAP2 activity, provided redundantly by two paralogs, is necessary for the maintenance of MSCs, and that BOFS-associated TFAP2A variants inhibit both paralogs, leading to ectopic differentiation of MSCs and to hair graying. We will test this hypothesis in mice by deleting the relevant Tfap2 paralogs in MSCs or in cells of the MSC niche, and examining hair graying and MSC status. In Aim 2 we will test the hypothesis that TFAP2 paralogs serve as pioneer factors at a subset of the loci to which MITF binds, facilitating MITF-mediated activation of genes that promote growth and differentiation. This will entail deleting TFAP2 paralogs from a melanoma cell line and then assessing open chromatin using ATAC- SEQ, and binding of MITF using ChIP-SEQ. We will carry out parallel experiments in primary melanoblasts isolated from zebrafish embryos. In Aim 3 we will test the hypothesis that a negative feedback loop in TFAP2A signaling is necessary for the correct balance of maintenance and recruitment of MSCs. We found that a potent inhibitor of TFAP2-paralog transactivation activity, KCTD15, is expressed in melanocytes, and that zebrafish kctd15a/b mutants exhibit supernumerary, ectopic melanocytes. We will test the model that Kctd15- mediated sumoylation of Tfap2a blocks its transactivation activity, testing sumoylation in vitro. We will test kctd15a for a role in controlling melanocyte differentiation by abrogating its expression in the melanocyte lineage. We will also test the prediction that inhibition of TFAP2 mediates the effects of KCTD15 in the melanocyte lineage. The expected outcome of the proposed research is a deeper understanding of pathways that regulate the balance of growth and differentiation of MSCs. An understanding of the mechanisms whereby MITF and TFAP2 proteins contribute to MSC maintenance, and of how these factors are regulated, will have a broad impact as they participate in mechanisms of aging and in the initiation and progression of melanoma.